3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (E.C. 4.1.3.5) catalyzes the formation of a key intermediate in the cholesterogenic and ketogenic pathways in a three step process (Miziorko, et al., (1977) J. Biol. Chem. 252, 1414-1420):
(1) EnzSH+acetyl-CoA.fwdarw.acetyl-SEnz+CoASH PA1 (2) acetoacetyl-CoA+acetyl-SEnz.fwdarw.EnzS-HMG-CoA PA1 (3) EnzS-HMG-CoA+H.sub.2 O.fwdarw.EnzSH+HMG-CoA
Distinct hepatic isozymes catalyze the synthesis of cholesterogenic and ketogenic intermediates (Clinkenbeard et al., (1975) J. Biol. Chem. 250, 3124-3135). As anticipated for the enzyme that catalyzes the first irreversible step in these metabolic pathways, HMG-CoA synthase has been implicated as a control point (Smith et al., (1988) J. Biol. Chem. 263, 18480-18487; Casals et al., (1992) Biochem. J. 283, 261-264; Quant et al., (1989) Biochem. J. 262, 159-164). These observations account for the recent interest in developing anti-steroidogenic and anti-isoprenyl agents that selectively target this enzyme (Omura et al., (1987) J. Antibiotics 40, 1356-1357).
Elements of the active site of this important enzyme have been identified (Miziorko, et al. (1985) Biochemistry 24, 3174-3179; Miziorko, et al., (1985) J. Biol. Chem. 260, 13513-13516; Vollmer et al., (1988) Biochemistry 27, 4288-4292; Miziorko et al., (1990) Biochim. Biophys. Acta 1041, 273278) in studies that relied on synthase isolated from an avian source. These synthase preparations have particular characteristics that render their use in drug targeting studies problematic. For example, an avian liver cytosolic synthase (Clinkenbeard, et al., 1975, supra) was found to be proteolytically cleaved. This would account for the differences in molecular weight, isoelectric point, and chromatographic properties that were observed for four different putative cytosolic protein species that, upon isolation, were found to catalyze the formation of HMG-CoA. Miziorko ((1985) Methods of Enzymology 110, p. 19-26, Ed. J. H. Law and Hans C. Rilling) noted that a crude preparation of HMG-CoA synthase isolated from chicken liver had only 20% activity after storage at 4.degree. C. for 24 hours.
HMG-CoA synthase is used to assay drugs believed to be efficacious in cholesterol reduction because of the pivotal role the synthase plays in the production of cholesterol. Similarly, one could use HMG-CoA synthase preparations to assay drugs thought to be capable of inhibiting isoprenylation of proteins, which may be a step in cancer metabolism. Investigation of putative anti-cholesterol and anti-isoprene drugs is handicapped by the quality of prior art native HMG-CoA synthase preparations. Additional investigation of synthase properties could be facilitated by application of recombinant DNA methodology to allow more convenient production of the enzyme and engineered variants. We previously documented the isolation of full length cDNA encoding avian liver HMG-CoA synthase (Kattar-Cooley et al., (1990) Arch. Biochem. Biophys. 283, 523-529). The genes for the rat mitochondrial and cytosolic HMG-CoA synthase have been analyzed by Ayte, et al., (1990, Proc. Natl. Acad. Sci. U.S.A. 87, 3874-3878). These workers expressed cDNA clones of the synthase gene in E. coli with a resulting specific activity of between 0.4 and 1.2 milliunits/mg. Hamster HMG-CoA synthase was cloned by Gil, et al. (Gil, et al., 1986, J. Biol. Chem. 261, 3710-3716.).